Heart failure is an enormous public health problem. This proposal presents an exciting new clinically translatable gene delivery method for the treatment of heart failure. Our technique allows for efficient isolation of the heart in vivo, coupled with the administration of a cocktail of endothelial permeabilizing agents with retrograde re-circulation of recombinant adeno associated virus (rAAV) in the coronary circulation. This technique is predicted to result in a dramatic increase in the transendothelial delivery of rAAV into the cardiac interstitium. In several rodent models, including the normal rat and the BIO 14.6 delta sarcoglycan deficient hamster, using an analogous technique, we have demonstrated exudation of viral vectors into the interstitium and stable, uniform therapeutic and marker transgene expression in skeletal muscle in situ and the heterotopically transplanted rodent heart. We have previously demonstrated global cardiac-specific transgene expression in the canine heart using cardiopulmonary bypass and antegrade vector delivery without the use of inflammatory mediators. In this proposal we develop a new cardiac surgical method allowing for isolation of the heart in situ with retrograde perfusion of the coronary circulation resulting in increased capillary and venular transendothelial pressure gradients. We also design a circuit for retrograde cardiac perfusion with minimum priming volume to further increase circulating vector concentration. Through isolation of the heart our approach uniquely allows for control of the ionic composition and temperature of the delivery vehicle and multiple passes of each rAAV vector through the heart. Based on our preliminary data and published results, each of these factors: retrograde perfusion, cardiac isolation, increased vector concentration, vector re-circulation, optimum temperature, ionic composition and the administration of endothelial permeabilizing agents is predicted to significantly improve the efficiency of rAAV-mediated cardiac gene delivery. We will also construct a novel reporter gene construct, rAAV- (MHC)-DAT, allowing for cardiac-specific gene expression and non-invasive quantitative assessment of gene expression using SPECT imaging. Our surgical method and perfusion circuit for isolated retrograde rAAV delivery to the myocardium offers several theoretical advantages over all other available delivery techniques. Further development of this approach may ultimately lead to the development of clinically relevant techniques of therapeutic cardiac gene delivery for the treatment of heart failure of diverse etiologies.